The present invention relates to a carboxylic acid derivative and a peroxisome proliferator activated receptor regulator containing carboxylic acid derivative as active ingredient.
More particularly, the present invention relates to a compound of the formula 
(wherein, all the symbols are the same meanings as defined hereafter), non-toxic salt thereof and hydrate thereof, process for producing the same and peroxisome proliferator activated regulator containing the same as an active ingredient.
Recently in the study of transcription factors concerned with marker genes expression in adipocytes differentiation, peroxisome proliferator activated receptor (abbreviated as PPAR hereinafter), which is one of intranucler receptors, has been focused. cDNAs of PPAR were cloned from various kinds of animals, and plural isoform genes were found, particularly in mammals three types of isoforms (xcex1, xcex4, xcex3) are known (see J. Steroid Biochem. Molec. Biol., 51, 157 (1994); Gene Expression,. 4, 281 (1995); Biochem Biophys. Res. Commun., 224, 431 (1996); Mol. Endocrinology., 6, 1634 (1992)). PPARxcex3 isoform is predominantly expressed in adipose tissues, immune cells, adrenal gland, spleen, small intestine. PPARxcex1 isoform is mainly expressed in adipose tissue, liver, retina, and PPARxcex4 isoform is widely expressed without specificity for tissue (see Endocrinology., 137, 354 (1996)).
On the other hand, the following thiazolidine derivatives are known as agents for the treatment of non-insulin dependent diabetes mellitus (NIDDM) and are hypoglycemic agents which are used for the improvement of hyperglycemia in the patients suffering from diabetes. They are also effective for the improvement of hyperinsulinemia, glucose tolerance and decrease of serum lipid and therefore they are thought to be considerably hopeful as agents for the treatment of insulin resistance. 
One of the target proteins in the cells of these thiazolidine derivatives is exactly PPARxcex3 and it is resolved that they enhance the transcription activity of PPARxcex3 (see Endocrinology., 137, 4189 (1996); Cell., 83, 803 (1995); Cell., 83, 813 (1995); J. Biol. Chem., 270, 12953 (1995)). Therefore, a PPARxcex3 activator (agonist) which enhances its transcription activity is thought to be hopeful as a hypoglycemic agent and/or a hypolipidemic agent. Furthermore, since a PPARxcex3 agonist is known to promote the expression of PPARxcex3 protein itself (Genes and Development., 10, 974 (1996)), an agent which increases the expression of PPARxcex3 protein itself as well as PPARxcex3 activating agent is also thought to be clinically useful.
Among all of nuclear receptors, PPARxcex3 is related to adipocytes differentiation (see J. Biol. Chem., 212, 5637 (1997) and Cell., 83, 803 (1995)). It is known that thiazolidine derivatives which activate this receptor promote adipocytes differentiation. Recently it was reported that thiazolidine derivatives increase fat mass and cause man to gain weight and to become obese (see Lancet., 349, 952 (1997)). Therefore, it is also thought that antagonists which inhibit PPARxcex3 activity and agents that decrease the expression of PPARxcex3 protein itself are also clinically applicable. On the other hand, a compound that phosphorylates PPARxcex3 protein and decreases its activity is reported (Science., 274, 2100 (1996)). This implies that an agent which does not bind on PPARxcex3 protein as a ligand, but inhibits its activity is also clinically applicable.
From these, PPARxcex3 activators (agonists) and PPARxcex3 regulators for its expression that can increase the expression of the protein itself are expected to be useful as hypoglycemic agents, hypolipidemic agents, and agents for prevention and/or treatment of diseases associated with metabolic disorders such as diabetes, obesity, syndrome X, hypercholesterolemia and hyperlipoproteinemia etc., hyperlipidemia, atherosclerosis, hypertension, circulatory diseases and overeating etc.
On the other hand, antagonists that inhibit the transcription activity of PPARxcex3 or PPARxcex3 regulators that inhibit the expression of the protein itself are expected to be useful as hypoglycemic agents and agents for prevention and/or treatment of diseases associated with metabolic disorders such as diabetes, obesity and syndrome X etc., hyperlipidemia, atherosclerosis, hypertension and overeating etc.
The following fibrate compound (e.g. chlofibrate) is known as a hypolipidemic agent. 
And, it is also resolved that one of the target proteins in the cells of fibrate compounds is PPARxcex1 (See Nature., 347, 645 (1990); J. Steroid Biochem. Molec. Biol., 51, 157 (1994); Biochemistry., 32, 5598 (1993)). From these facts, PPARxcex1 regulators which can be activated by fibrate compounds are thought to have a hypolipidemic effect, and so they are expected to be useful as agents for prevention and/or treatment of hyperlipidemia etc.
Besides, it has been recently reported that PPARxcex1 possesses anti-obese activity in the specification of WO 9736579. In addition, it was reported that the elevation of high density lipoprotein (HDL) cholesterol level and the reduction of low density lipoprotein (LDL) cholesterol, very low density lipoprotein (VLDL) cholesterol and triglyceride levels were induced by activation of PPARxcex1 (J. Lipid Res., 39, 17 (1998)). It was also reported that composition of fatty acids in blood, hypertension and insulin resistance were improved by administration of bezafibrate which is one of bezafibrate compounds (Diabetes., 46, 348 (1997)).
Therefore, agonists that activate PPARxcex1 and PPARxcex1 regulators that promote expression of PPARxcex1 protein itself are useful as hypolipidemic agents and agents for treatment of hyperlipidemia, and are expected to have HDL cholesterol level-elevating effect, LDL cholesterol and/or VLDL cholesterol levels-lowering effect, inhibition on the progress of atherosclerosis and anti-obese effect. Therefore, they are thought to be hopeful agents for the treatment and/or prevention of diabetes as hypoglycemic agents, for the improvement of hypertension, for the relief from risk factor of syndrome X and for the prevention of occurrence of ischemic coronary diseases.
On the other hand, few reports are found on ligands that activate PPARxcex4 significantly or on biological activities associated with PPARxcex4
PPARxcex4 is sometimes called PPARxcex2, or it is also called NUC1 in human. Until now, as for activity of PPARxcex4, it is disclosed in the specification of WO 9601430 that hNUC1B (PPAR subtype whose structure is different from that of human NUC1 in one amino acid) inhibited the transcription activities of human PPARxcex1 and thyroid hormone receptor. Recently in the specification of WO 9728149, it was reported that the compounds, which possessed high affinity to PPARxcex4 protein and which could activate PPARxcex4 significantly (i.e. agonists) were found out and that they had HDL (high density lipoprotein) cholesterol level-elevating activity. Therefore, agonists that can activate PPARxcex4 are expected to have HDL cholesterol level-elevating effect, and so they are expected to be useful for the inhibition on the progress of atherosclerosis and treatment thereof, as hypolipidemic agents and hypoglycemic agents, for the treatment of hyperlipidemia, as hypoglycemic agents, for the treatment of diabetes, for the relief from risk factor of syndrome X, and for the prevention of occurrence of ischemic coronary diseases.
As for PPAR regulators, the following compounds were reported besides the above-mention thiazolidine derivatives and fibrate compounds.
For example, in WO9731907, it is disclosed that the compounds of the formula (A) 
(wherein, AA is phenyl, in which the said phenyl may be substituted with one or more substituent(s) selected from group consisting of halogen, C1-6 alkyl, C1-3 alkoxy, C1-3 fluoroalkoxy, nitrile or xe2x80x94NR7AR8A (R7A and R8A each independently, is hydrogen or C1-3 alkyl);
BA is (5- or 6-membered heterocyclic ring containing at least one hetero atom selected from O, N and S)-C1-6 alkylene-, in which the said heterocyclic ring may be substituted with C1-3 alkyl;
AlkA is C1-3 alkylene;
R1A is hydrogen or C1-3 alkyl;
ZA is xe2x80x94(C1-3 alkylene)phenyl or xe2x80x94NR3A R4A)
or pharmaceutically acceptable salts thereof possess PPARxcex3 agonist activity (the necessary parts in explanation of symbols are shown).
On the other hand, in JP-A-9-323982, it is disclosed that the propionic acid derivatives of the formula (B) 
(wherein, R8 is 
(wherein, R8 is substituted or unsubstituted aromatic hydrocarbon, substituted or unsubstituted aliphatic hydrocarbon ring, substituted or unsubstituted heterocyclic ring or substituted or unsubstituted condensed heterocyclic ring, R5B is lower alkyl), R4B is hydrogen or lower alkyl, R6B is hydrogen or R6B and R9B taken together form double bond, R7B is hydrogen, hydroxy, carboxy, acyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted lower alkyl, substituted or unsubstituted carbamoyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted aralkyloxycarbonyl or xe2x80x94YBxe2x80x94R8B (in which YB is xe2x80x94NHxe2x80x94 or O, R8B is substituted or unsubstituted acyl, substituted or unsubstituted alkoxycarbonyl, aryloxycarbonyl or aralkyloxycarbonyl), R9B is hydrogen, substituted or unsubstituted lower alkyl or substituted or unsubstituted lower alkoxycarbonyl, R10B is hydroxy, substituted or unsubstituted amino, substituted or unsubstituted lower alkoxy, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryloxy or substituted or unsubstituted aralkyloxy) or pharmaceutically acceptable salts thereof possess hypoglycemic action and hypolipidemic action. In addition, JP-A-8-325264, JP-A-8-325250, WO9638415 and WO9800137 have also disclosed that analogous compounds possess hypoglycemic action and hypolipidemic action.
As the result of energetic investigation in order to find compounds possessing regulating action on PPAR, the present inventors have found that the purpose has been accomplished by the compound of the formula (I) and have completed the present invention.
The present invention relates to,
(1) a compound of the formula (I) 
(wherein,
A1 is C1-4 alkylene,
A2 is C2-8 alkylene, C3-8 alkenylene or C3-8 alkynylene (with the proviso that (a) when E2 is xe2x80x94Oxe2x80x94 or xe2x80x94S(O)mxe2x80x94, A2 represent neither C3 alkenylene nor C3 alkynylene and (b) each carbon atom in A2 which is connected to E1 and E2 in which E2 is Oxe2x80x94 or xe2x80x94S(O)mxe2x80x94 does not possess double bond),
E1 is xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94,
E2 is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94S(O)mxe2x80x94,
m is 0, 1 or 2,
each R1 in (R1)n independently, is hydrogen, C1-8 alkyl, halogen, C1-4 alkoxy, C1-4 alkylthio, nitro, NR7R8 (in which R7 and R8 each independently, is C1-4 alkyl), cyano, trifluoromethyl, trifluoromethyloxy, carbocyclic ring or heterocyclic ring (in which carbocyclic ring and heterocyclic ring may be substituted with the group selected from C1-4 alkyl, C1-4 alkoxy, halogen or trifluoromethyl),
R2 is hydrogen, C1-8 alkyl, halogen, C1-4 alkoxy, C1-4 alkylthio, nitro, NR7R8 (in which R7 and R8 each independently, is C1-4 alkyl), cyano, trifluoromethyl or trifluoromethyloxy,
R3 and R4 each independently, is hydrogen or C1-4 alkyl or R3 and R4 taken together with carbon atom to which is attached represents C3-7 cycloalkylene,
R5 is xe2x80x94COOR9 (in which R9 is hydrogen or C1-4 alkyl) or heterocyclic ring which is equivalent to carboxylic acid, 
each independently, is carbocyclic ring or heterocyclic ring and n is 1-3)
or non-toxic salt thereof, or hydrate thereof,
(2) a peroxisome proliferator activated receptor regulator comprising, as an active ingredient, a compound of the formula (I) or non-toxic salt thereof, or hydrate thereof, and
(3) a process for producing a compound of the formula (I).
Unless otherwise specified, all isomers are included in the present invention. For example, alkyl, alkoxy, alkylthio, alkylene, alkenylene and alkynylene group includes straight or branched ones. In addition, isomers on double bond, ring, fused ring (E-, Z-, cis-, trans-isomer), isomers generated from asymmetric carbon atom(s) (R-, S-, xcex1-, xcex2-isomer, enantiomer, diastereomer), optically active isomers (D-, L-, d-, I-isomer), polar compounds generated by chromatographic separation (more polar compound, less polar compound), equilibrium compounds, mixtures thereof at voluntary ratios and racemic mixtures are also included in the present invention.
In the formula (I), C1-4 alkylene represented by A1 means methylene, ethylene, trimethylene, tetramethylene and isomers thereof.
In the formula (I), C1-4 alkyl represented by R3, R4, R7, R8 and R9 or C1-4 alkyl as a substituent of carbocyclic ring or heterocyclic ring represented by R1 means methyl, ethyl, propyl, butyl and isomers thereof.
In the formula (I), C1-8 alkyl represented by R1 and R2 means methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl and isomers thereof.
In the formula (I), C1-4 alkoxy represented by R1 and R2 or C1-4 alkoxy as a substituent of carbocyclic ring or heterocyclic ring represented by R1 means methoxy, ethoxy, propoxy, butoxy and isomers thereof.
In the formula (I), C1-4 alkylthio represented by R1 and R2 means methylthio, ethylthio, propylthio, butylthio and isomers thereof.
In the formula (I), C2-8 alkylene represented by A2 means ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene,heptamethylene, octamethylene and isomers thereof.
In the formula (I), C3-8 alkenylene represented by A2 means 1-propynylene, 1-butenylene, 2-butenylene, 2-methyl-2-butenylene, 3-methyl-2-butenylene, pentenylene, hexenylene, heptenylene, octenylene and isomers thereof.
In the formula (I), C3-8 alkynylene represented by A2 means 1-propynylene, 1-butynylene, 2-butynylene, pentynylene, hexynylene, heptynylene, octynylene and isomers thereof.
In the formula (I), halogen represented by R1 and R2 or halogen as a substituent of carbocyclic ring or heterocyclic ring represented by R1 means fluoride, chloride, bromide and iodide.
In the formula (I), C3-7 cycloalkylene represented by R3 and R4 taken together with carbon atom to which is attached means cycloheptylene, cyclobutylene, cyclopentylene, cyclohexylene and cycloheptylene.
In the formula (I), heterocyclic ring which is equivalent to the carboxylic acid represented by R5 includes, for example, 1H-tetrazol-5yl, thiazolidine-2,4-dion-5-yl, oxazolidin-2,4-dion-5-yl, isooxazolidin-3,5-dion-4-yl, 1,2,4-oxadiazolidin-3,5-dion-2-yl etc. shown by the following structure: 
but does not limit to the said group.
In the formula (I), carbocyclic ring represented by R1, 
means C3-10 mono- or bi-cyclic carbocyclic ring and bicyclo carbocyclic ring. For example, C3-10 mono- or bi-cyclic carbocyclic ring and bicyclo carbocyclic ring mean cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, cyclopentene, cyclohexene, cyclopentadien, cyclohexadien, benzene, pentalene, indene, naphthalene, azulene, dihydronaphthalene, tetrahydronaphthalene, perhydronaphthalene, indane (dihydroindene), perhydroindene, bicyclopentane, bicyclohexane, bicycloheptane ([2.2.1]bicycloheptane), bicyclooctane, bicyclononane, bicyclodecane, adamantane etc.
In the formula (I), heterocyclic ring represented by R1, 
means 5-15-membered mono- or bi-cyclic heterocyclic ring containing 1-3 nitrogen atom(s), 1-2 oxygen atom(s) and/or one sulfur atom which is unsaturated or saturated partially or fully, For example, 5-15-membered mono- or bi-cyclic heterocyclic ring containing 1-3 nitrogen atom(s), 1-2 oxygen atom(s) and/or one sulfur atom which is unsaturated or saturated partially or fully includes pyroline, pyrrolidine, imidazoline, imidazolidine, pyrazoline, piperazolidine, piperidine, piperazine, tetrahydropyrimidine, hexahydropyrimidine, tetrahydropyridazine, hexahydropyridazine, hexahydroazepine, dihydrofuran, tetrahydrofuran, dihydropyran, tetrahydropyran, dihydrothiophene, tetrahydrothiophene, dihydrothiain (dihydrothiopyran), tetrahydrothiain (tetrahydrothi opyran), dihydrooxazole, tetrahydrooxazole, dihydroisooxazole, tetrahydroisooxazole, dihydrothiazole, tetrahydrothiazole, dihydroisothiazole, tetraisothiazole, morpholine, thiomorpholine, indoline, isoindoline, dihydroindazole, perhydroindazole, dihydroquinoline, tetrahydroquinoline, perhydroquinoline, dihydroisoquinoline, tetrahydroisoquinoline, perhydroisoquinoline, dihydrophthalazine, tetrahydrophthalazine, perhydrophthalazine, dihydronaphthylidine, tetrahydronaphthylidine, perhydronaphthylidine, dihydroquinoxaline, tetrahydroquinoxaline, perhydroquinoxaline, dihydroquinazoline, tetrahydroquinazoline, perhydroquinazoline, dihydrocinnoline, tetrahydrocinnoline, perhydrocinnoline, dihydrobenzooxazole, perhydrobenzooxazole, dihydrobenzothiazole, perhydrobenzothiazole, dihydrobenzoimidazole, perhydrobenzoimidazole, dihydrobenzooxazine, dioxaindane (1,3-dioxaindane), benzodioxane, quinuclidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, pyridazine, azepine, diazepine, furan, pyran, oxepine, oxazepine, thiophene, thiain (thiopyran), thiepine, oxazole, isooxazole, thiazole, isothiazole, oxadiazole, oxazine, oxadiazine, oxazepine, oxadiazepine, thiadiazole, thiazine, thiadiazine, thiaazepine, thiaazepine, thiadiazepine, indole, isoindole, benzofuran, isobenzofuran, benzothiophene, isobenzothiophene, indazole, quinoline, isoquinoline, phthalazine, naphthylidine, quinoxaline, quinazoline, cinnoline, benzooxazole, benzothiazole, benzoimidazole, oxatetrahydrofuran, imidazopyridine, benzotriazole etc.
In the formula (I), A1 is preferably, every group and more preferably methylene or ethylene.
In the formula (I), A2 is preferably, every group, more preferably C4-6 alkylene, C4-6 alkenylene, C4-6 alkynylene, much more preferably tetramethylene 2-butenylene, 2-butynylene, or the said group which is substituted with 1-2 of methyl, and most preferably 2-butynylene.
In the formula (I), E1 is preferably, every group and more preferably xe2x80x94Oxe2x80x94.
In the formula (I), E2 is preferably, every group and more preferably xe2x80x94Sxe2x80x94 or xe2x80x94CH2xe2x80x94.
In the formula (I), R5 is preferably, every group, more preferably xe2x80x94COOR9 , 1-tetrazol-5-yl, or thiazolidine-2,4-dion-5-yl and most preferably xe2x80x94COOR9.
In the formula (I), 
is preferably, every group, more preferably C5-6 mono-cyclic carbocyclic ring, or 5-6-membered mono-cyclic heterocyclic ring containing 1-2 nitrogen atom(s), one oxygen atom and/or one sulfur atom, much more preferably 
and most preferably 
In the formula (I), 
is preferably, every group, more preferably C5-10 mono- or bi-cyclic carbocyclic ring, or 5-1 0-membered mono- or bi-cyclic heterocyclic ring containing 1-2 nitrogen atom(s), 1-2 oxygen atom(s) and/or one sulfur atom which is unsaturated or saturated partially or fully, and most preferably cyclopentane, cyclohexane, cycloheptane, benzene, furan, thiophene, pyridine, quinoline, dioxaindane (for example, 1,3-dioxaindane).
In the present invention, PPAR regulator includes all the regulators of PPARxcex1, xcex3, xcex4, xcex1+xcex3, xcex1+xcex4 and xcex1+xcex3+xcex4. Preferable regulatory fashion is, PPARxcex1 regulator, PPARxcex3 regulator, PPARxcex4 regulator, PPARxcex1+xcex3 regulator, PPARxcex1+xcex4 regulator, more preferably PPARxcex1+xcex3 regulator.
PPAR regulator also includes PPAR agonist and PPAR antagonist, preferably PPAR agonist, more preferably PPARxcex1 agonist, PPARxcex3 agonist, PPARxcex4 agonist, PPARxcex1+xcex3 agonist or PPARxcex1+xcex4 agonist, particularly preferably PPARxcex1+xcex3 agonist.
Among the compounds of the formula (I), compounds of the formula (Ia) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ib) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ic) 
(wherein, all the symbols are the same meanings as defined hereinbefore), non-toxic salt thereof, or hydrate thereof are preferable.
The compounds of the formula (Ia-A) 
(wherein, A2-3 is C3-8 alkynylene and the other symbols are the same meanings as defined hereinbefore), compounds of the formula (Ia-B) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ia-C) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ib-A) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ib-B) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ib-C) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ic-A) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ic-B) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ic-C) 
(wherein, all the symbols are the same meanings as defined hereinbefore), non-toxic salt thereof, or hydrate thereof are more preferable.
The compounds of the formula (Ia-1) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ia-2) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ia-3) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ia-4) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ia-5) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ia-6) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ib-1) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ib-2) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ib-3) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ic-1) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ic-2) 
(wherein, all the symbols are the same meanings as defined hereinbefore), compounds of the formula (Ic-3) 
(wherein, all the symbols are the same meanings as defined hereinbefore), non-toxic salt thereof, or hydrate thereof are most preferable.
Concrete compounds of the present invention are, for example, the compounds shown in the following Tables 1-16, non-toxic salts thereof and hydrates thereof, and compounds described in Example hereafter.
In each Table, Me is methyl and the other symbols are the same meanings as defined hereinbefore.
(a) Compounds of the formula (I) wherein E2 is xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94 and R5 is xe2x80x94COOR9-1 (in which R9-1 is C1-4 alkyl) or heterocyclic ring which is equivalent to carboxylic acid, i.e., the compounds of the formula (I-A) 
(wherein, E2-1 is xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, R5-1 is xe2x80x94COOR9-1 (in which R9-1 is C1-4 alkyl) or heterocyclic ring which is equivalent to carboxylic acid and the other symbols are the same meanings as defined hereinbefore)
may be prepared by reacting the compounds of the formula (II) 
(wherein, R10 is methanesulfonyloxy or halogen and the other symbols are the same meanings as defined hereinbefore) and the compounds of the formula (III) 
(wherein, E2-1a is xe2x80x94OH or xe2x80x94SH and the other symbols are the same meanings as defined hereinbefore).
The said reaction may be carried out by known methods. It may be carried out, for example, in an organic solvent (tetrahydrofuran (THF), diethylether, methylene chloride, chloroform, tetrachloromethane, pentane, hexane, benzene, toluene, dimethylformamide (DMF), dimethylsulfoxide (DMSO), hexamethylphosphamide (HMPA), acetonitrile etc.) in the presence of base (sodium hydroxide, potassium carbonate, triethylamine, pyridine, sodium iodide, potassium iodide, cesium carbonate etc.) at 0-80xc2x0 C.
(b) Compounds of the formula (I) wherein E2 is xe2x80x94CH2xe2x80x94 and R5 is xe2x80x94COOR9-1 (in which R9-1 is C1-4 alkyl) or heterocyclic ring which is equivalent to carboxylic acid, i.e., the compounds of the formula (I-B) 
(wherein, E2-2 is xe2x80x94CH2xe2x80x94 and the other symbols are the same meanings as defined hereinbefore)
may be prepared by reacting the compounds of the formula (IV) 
(wherein, all the symbols are the same meanings as defined hereinbefore) and the compounds of the formula (V-1) 
(wherein, E1-1 is xe2x80x94OH or xe2x80x94SH and the other symbols are the same meanings as defined hereinbefore) or by reacting the compounds of the formula (VII) 
(wherein, all the symbols are the same meanings as defined hereinbefore) and the compounds of the formula (V-2) 
(wherein, all the symbols are the same meanings as defined hereinbefore).
The said reaction may be carried out by known methods. It may be carried out, for example, in an organic solvent (THF, diethylether, methylene chloride chloroform, tetrachloromethane, pentane, hexane, benzene, toluene, DMF, DMSO, HMPA, or mixture solvent thereof etc.) in the presence of base (sodium hydroxide, potassium carbonate, triethylamine, pyridine, sodium iodide, cesium carbonate etc.) at 0-80xc2x0 C.
(c) Compounds of the formula (I) wherein E2 is xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, or xe2x80x94CH2xe2x80x94and R5 is xe2x80x94COOH, i.e., the compounds of the formula (I-C) 
(wherein, E2-3 is xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, or xe2x80x94CH2xe2x80x94 and the other symbols are the same meanings as defined hereinbefore) may be prepared by hydrolysis of the compounds of the formula (I-A) wherein R5-1 is xe2x80x94COOR9-1, i.e., the compounds of the formula (I-A-1) 
(wherein, all the symbols are the same meanings as defined hereinbefore) or the compounds of the formula (I-B) wherein R5-1 is xe2x80x94COOR9-1, i.e., the compounds of the formula (I-B-1) 
(wherein, all the symbols are the same meanings as defined hereinbefore).
In addition, compounds of the formula (I-C) wherein A2 is C3-8 alkynylene and E2 is xe2x80x94CH2xe2x80x94, i.e., the compounds of the formula (I-C-1) 
(wherein, A2-3 is C3-8 alkynylene and the other symbols are the same meanings as defined hereinbefore)
may be prepared by hydrolysis of the compounds of the formula (XII) 
(wherein, R6 is a carboxyl group which is protected (for example, 4-methyl-2,6,7-trioxabicyclo[2,2,2]octan-1-yl etc.) and the other symbols are the same meanings as defined hereinbefore).
The said hydrolysis may be carried out by known methods. It may be carried out, for example,
(1) in an organic solvent admissible with water (THF, dioxane, ethanol, methanol etc.) or mixture solvent thereof, using an aqueous solution of alkali (potassium hydroxide, sodium hydroxide, lithium hydroxide, potassium carbonate, sodium carbonate etc.),
(2) in alkanol (methanol, ethanol etc.), using the above alkali under an anhydrous condition or
(3) in an organic solvent (THF, dioxane, ethanol, methanol etc.) or mixture solvent thereof, using acid (hydrochloric acid, sulfuric acid etc.) and alkali (sodium hydroxide, potassium hydroxide etc.).
These reactions may be carried out at 0-100xc2x0 C. normaly. In addition, the said hydrolysis may be carried out
(4) in mixture solvent of an organic solvent admissible with water (ethanol, dimethylsulfoxide etc.) and water, in the presence or absence of buffer, using enzyme to decompose ester (esterase, lipase etc.) at 0-50xc2x0 C.
(d) Compounds of the formula (I) wherein E2 is xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94CH2xe2x80x94 and heterocyclic ring represented by R5 which is equivalent to carboxylic acid is 1H-tetrazol-5-yl, i.e., the compounds of the formula (I-D) 
(wherein, all the symbols are the same meanings as defined hereinbefore) may be also prepared by reacting the compounds of the formula (VI) 
(wherein, all the symbols are the same meanings as defined hereinbefore) in an alkaline condition.
The said reaction may be carried out by known methods. It may be carried out, for example, in an organic solvent admissible with water (methanol, ethanol, dioxane etc.), using alkali (potassium hydroxide, sodium hydroxide, potassium carbonate, sodium carbonate etc.) at 0-50xc2x0 C.
(e) Compounds of the formula (I) wherein m is 1 or 2, that is to say, E2 is xe2x80x94SOxe2x80x94 or xe2x80x94SO2xe2x80x94, i.e., the compounds of the formula (I-E) 
(wherein, E2-4 is xe2x80x94S(O)mm-(in which mm is 1 or 2) and the other symbols are the same meanings as defined hereinbefore)
may be prepared by oxidizing the compounds of the formula (I-A) wherein E2-1 is xe2x80x94Sxe2x80x94 or the compounds of the formula (I-C) or (I-D) wherein each E 2-3 is xe2x80x94Sxe2x80x94.
In addition, the compounds of the formula (I-E) wherein E 2-4 is xe2x80x94SO2xe2x80x94 may be also parepared by oxidizing the compounds of the formula (I-E) wherein E2-4 is xe2x80x94SOxe2x80x94.
The said oxidizing may be carried out by known methods. It may be carried out, for example in an organic solvent (THF, methylene chloride, chloroform etc.), using a necessaryl amount of oxidizing agent (perhydrogen oxide, sodium periodate, acyl nitrite, sodium perboronate, peracid (e.g., 3-chloroperbenzoic acid, peracetic acid, OXONE (trade mark)) etc.) at 0-50xc2x0 C.
The compounds of the formulae (II), (III), (IV), (V-1), (V-2), (VI), (VII) and (XII) are known per se or may be prepared by known methods.
For example, among the compounds of the formula (III), mercaptoacetic acid methyl ester (thioglycolic acid methyl) has been marketed.
For example, the compounds of the formulae (II), (IV), (VI) and (XII) may be prepared according to the following Reaction Schemes 1-4.
In each Reaction Scheme, the abbrevation and symbols are as the following meanings and the other symbols are the same meanings as defined hereinbefore.
THP: tetrahydropyran-2-yl,
A2-1: C3-8 alkenylene or C3-8 alkynylene,
X: halogen,
A2-2: C2-8 alkylene,
EDC HCl: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride,
HOBt: 1-hydroxybenzotriazole,
Et3N: triethylamine,
TMSN3: trimethylsilylazide. 
In the said Reaction Schemes, the compounds of the formula (VII), (VIII-1), (VIII-2), (XI) and (XVII) are known per se or may be prepared by known methods.
The reactions described in the above-mentioned Schemes may be carried out by known methods.
In the present invention, the other starting materials and each reagent are known per se or may be prepared by known methods.
In each reaction in the present specification, products may be purified by a conventional manner. For example, it may be carried out by distillation at atmospheric or reduced pressure, high performance liquid chromatography, thin layer chromatography or column chromatography using silica gel or magnesium silicate, washing or recrystallization. Purification may be carried out after each reaction or after a series of reactions.
All the non-toxic salts are also included in the present invention. For example, the compounds of the formula (I) of the present invention may be converted into the corresponding salts by known methods. Non-toxic and water-soluble salts are preferable. Suitable salts, for example, are follows: salts of alkaline metals (potassium, sodium etc.), salts of alkaline earth metals (calcium, magnesium etc.), ammonium salts, salts of pharmaceutically acceptable organic amines (tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclopentylamine, cyclohexylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris(hydroxymethyl)amine, lysine, arginine, N-methyl-D-glucamine etc.).
The compounds of the formula (I) of the present invention may be converted into the corresponding acid additional salts by methods known per se. Non-toxic and water-soluble acid addition salts are preferable. Suitable acid addition salts, for example, are salts of inorganic acids, e.g., hydrochloride, hydrobromide, sulphate, phosphate, nitrate etc., or salts of organic acids, e.g., acetate, trifluoroacetate, lactate, tartarate, oxalate, fumarate, maleate, citrate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, toluenesulphonate, isethioate, glucuronate, gluconate etc.
The compounds of the formula (I) of the present invention or salts thereof may be converted into hydrate thereof by methods known per se.
It was confirmed that a compound of the present invention of formula (I) has PPAR regulating activities by the following experiments.
Measurement of PPARxcex1 Agonistic and PPARxcex3 Agonistic Activities
(1) Preparation of Materials in Luciferase Assay Using Human PPARxcex1 or xcex4
The whole operations were carried out by the basic methods in gene engineering techniques and the conventional methods in yeast One-hybrid or Two-hybrid system.
As a luciferase gene expression vector under the control of thymidine kinase (TK) promotor, luciferase structural gene was exiced from PicaGene Basic Vector 2 (trade name, Toyo Ink Inc., catalogue No. 309-04821), to prepare luciferase gene expression vector pTK-Luc. under the control of TK promotor (xe2x88x92105/+51) as a minimum essential promotor activity from pTKxcex2 having TK promotor (Chrontech Inc., catalogue No. 6179-1). In the upper stream of TK promotor, four times repeated UAS sequence was inserted, which is the response element of Gal4 protein, a basic transcription factor in yeast, to construct 4xc3x97UAS-TK-Luc. as reporter gene. The following is the enhancer sequence used (Sequence No. 1).
Sequence No. 1: Enhancer sequence repeating Gal4 response element four-times tandemly.
5xe2x80x2-T(CGACGGAGTACTGTCCTCCG)xc3x974 AGCT-3xe2x80x2
A vector was prepared as described hereafter which expresses chimeric receptor protein wherein in carboxyl terminus of yeast Gal4 protein DNA binding domain was fused to ligand binding domain of human PPARxcex1 or xcex3. That is to say, PicaGene Basic Vector 2 (trade name, Toyo Ink Inc., catalogue No. 309-04821) was used as a basic expression vector, the structural gene was exchanged for that of chimeric receptor protein, while promotor and enhancer domains were kept as they were.
DNA encoding a fused protein composed of Gal4 DNA binding domain, the 1st to 147th amino acid sequence linked to the ligand binding domain of human PPARxcex1 or xcex3 in frame was inserted to the downstream of promotor/enhancer in PicaGene Basic Vector 2 (trade name, Toyo Ink Inc., catalogue No. 309-04821). Here the DNA was aligned as follows; in the amino terminus of human PPARxcex1 or xcex3 ligand binding domain, nuclear translocation signal originated from SV-40 T-antigen, Ala Pro Lys Lys Lys Arg Lys Val Gly (sequence No. 2) was added to make fusion protein localizing intranuclearly. On the other hand, in the carboxy terminus of them, influenza hemagglutinin epitope, Tyr Pro Tyr Asp Val Pro Asp Tyr Ala (sequence No. 3) and stop codon for translation was added in this order, to detect an expressed fused protein tagged epitope sequence.
According to the comparison of human PPAR structures described in the literature by R. Mukherjee at al. (See J. Steroid Biochem. Molec. Biol., 51, 157 (1994)), M. E. Green et al., (See Gene Expression., 4, 281 (1995)), A. Elbrecht et al. (See Biochem Biophys. Res. Commun., 224, 431 (1996)) or A. Schmidt et al. (See Mol. Endocrinology., 6, 1634 (1992)), the portion of structural gene used as ligand binding domain of human PPARxcex1 or xcex3 was DNA encoding the following peptide:
human PPARxcex1 ligand binding domain: Ser167-Tyr468 
human PPARxcex3 ligand binding domain: Ser176-Tyr478 
(each human PPARxcex31 ligand binding domain and human PPARxcex32 ligand binding domain is Ser204-Tyr506 which is identical sequence each other).
In order to measure basal level of transcription, an expression vector containing DNA binding domain of Gal4 protein lacking in PPAR ligand binding domain, which is exclusively encoding the 1st to 147th amino acid sequence in Gal4 protein was also prepared.
(2) Luciferase Assay Using Human PPARxcex1 or xcex3
CV-1 cells used as host cells were cultured by a conventional technique. That is to say, Dulbecco""s modified Eagle medium (DMEM) supplemented 10% bovine fetal serum (GIBCO BRL Inc., catalogue No. 26140-061) and 50 U/ml of penicillin G and 50 xcexcg/ml of streptomycin sulfate were used to culture CV-1 cells under the atmosphere of 5% carbon dioxide gas at 37xc2x0 C.
2xc3x97106 cells were seeded in a 10 cm dish, and once washed with the medium without serum, followed by addition of the medium (10 ml) thereto. Reporter gene (10 xcexcg), Gal4-PPAR expression vector (0.5 xcexcg) and 50 xcexcg of LipofectAMINE (GIBRO BRL Inc., catalogue No. 18324-012) were well mixed and added to the culture to introduce these DNAs into the host cells. They were cultured at 37xc2x0 C. for 5-6 hours, and thereto was added 10 ml of medium containing 20% of dialyzed bovine fetal serum (GIBRO BRL Inc., catalogue No. 26300-061), and then cultured at 37xc2x0 C. overnight. The cells were dispersed by trypsin, and they were again seeded in 96-well plates in a density of 8000 cells/ 100 ml of DMEM-10% dialyzed serum/well. Several hours after the cultivation, when cells were attached to the plastic ware, then 100 xcexcl of DMEM-10% dialyzed serum containing the compounds of the present invention, whose concentration is twice as high as the final concentration of them, was added thereto. The culture was settled at 37xc2x0 C. for 42 hours and the cells were dissolved to measure luciferase activity according to manufacturer""s instruction.
As to PPARxcex1 agonistic activity, the relative activity of the compounds of the present invention (10 xcexcM) was shown in Table 17, under the condition that luciferase activity was defined as 1.0 in case of carbacyclin (10 xcexcM) as a positive control compound, which could activate transcription of luciferase gene significantly to PPARxcex1 (See Eur. J. Biochem., 233, 242 (1996); Genes and Development., 10, 974 (1996)).
As to PPARxcex3 agonistic activity, the relative activity of the compounds of the present invention (10 xcexcM) was shown in Table 18, under the condition that luciferase activity was defined as 1.0 in case of troglitazone (10 xcexcM) as a positive control compound, which could activate transcription of luciferase gene significantly to PPARxcex3 (See Cell., 83, 863 (1995); Endocrinology., 137, 4189 (1996) and J. Med. Chem., 39, 665 (1996)) and has been already launched as hypoglycemic agent.
Furthermore, assay of each compound was carried out three times to examine its reproducibility and to confirm the dose dependent activity.
Hypoglycemic and Hypolipidemic Effects
Male, 7-weeks old KKAy/Ta mice weighed from 35 to 40 g (five mice per group) were pre-breaded for approximately one week and acclimatized for three days on milled diet. On the first day of the experiment (Day 0), mice were divided into some groups according to weight, plasma glucose and triglyceride (TG) levels to minimize the differences among groups. From the next day for two days they were given compounds by food mixture containing 0.03% (w/w) of the compound of the present invention or by milled diet only. At 13:00 of the third day, blood samples were collected to measure glucose and TG levels. The results are shown in Table 19. Additionally, there was no significant difference in the food intake between control group (milled diet only) and compounds-treated group (milled diet containing 0.03% compounds).
Hypocholesterolemic and Hypolipidemic Effects
Male, six-weeks old SD rats (five rats per group) were left to take milled diet and water ad libitum and were acclimatized for 1 week.
At 9:00 on the first day of the experiment (Day 0), blood sampling was done from tail vain. The rats were divided into some groups according to body weight, triglyceride (TG), non-esterified fatty acid (NEFA) and total cholesterol (TC) levels to minimize differences of the parameters among the groups. At 17:00 of the day, the compound of the present invention dissolved in 0.5% aqueous solution of carboxymethylcellulose (CMC) was orally administered, and thereafter, with hypercholesterolemic food (5.5% peanut oil, 1.5% cholesterol and 0.5% cholic acid were mixed with milled CRF-1 diet, Charles River Inc.) was given to the rats.
At 9:00 of the next day, blood sampling was done from tail vein. The lipid levels in blood (TG, NEFA and TC levels) after administration of the compounds of the present invention were measured. The results are shown in Table 20. There was no significant difference of the food intake between the control group (provided only 0.5% CMC) and the group treated with the compounds of the present invention.
The hypoglycemic or hypolipidemic effects observed in KKAy mice imply the possibility of preventives and/or remedies for diabetes and hyperlipidemia etc. Cholesterol-lowering and free fatty acid-lowering effects observed in high cholesterol diet-fed rats imply that the compounds of the present invention are useful as preventives and/or remedies of atherosclerosis etc.
The compounds of the present invention possess the hypoglycemic or hypolipidemic (TG, NEFA) effects as well as cholesterol-lowering effect, so they are expected to be more useful to compare with the marketed hypoglycemic or hypolipidemic drugs.
In addition, it has been known that hyperlipidemia, obesity or diabetes are one of cause of uncondition in liver function and hyperlipid in liver. Therefore, the compounds of the present invention are expected to be the drugs to improve liver function which has not been marketed.
The compounds of the formula (I) of the present invention, non-toxic salts thereof, acid addition salts thereof and hydrates thereof have PPAR regulating effect, and therefore are expected to be applied as hypoglycemic agents, hypolipidemic agents, preventives and/or remedies for diseases associated with metabolic disorders (diabetes, obesity, syndrome X, hypercholesterolemia and hyperlipoproteinemia etc.), hyperlipidemia, atherosclerosis, hypertension, circulatory diseases, overeating, coronary heart diseases etc., HDL cholesterol-elevating agents, LDL cholesterol and/or VLDL cholesterol-lowering agents and agents for relieving risk factors of diabetes or syndrome X.
The compounds of formula (I) of the present invention, non-toxic salts thereof, acid addition salts thereof and hydrates thereof have particularly PPARxcex1 agonist and/or PPARxcex3 agonist effect, and therefore are thought to be useful as hypoglycemic agents, hypolipidemic agents, preventives and/or remedies for diseases associated with metabolic disorders (diabetes, obesity, syndrome X, hypercholesterolemia, hyperlipoproteinemia etc.), hyperlipidemia, atherosclerosis, hypertension, circulatory diseases and overeating etc. Since they are expected to have HDL cholesterol-elevating effect, LDL cholesterol and/or VLDL cholesterol-lowering effect, inhibition of progress of atherosclerosis and its treatment, and inhibitory effect against obesity, they are also expected to be useful for the treatment and/or prevention of diabetes as hypoglycemic agents, for the amelioration of hypertension, for the relief from risk factors of syndrome X, and as preventives against occurrence of coronary heart diseases.
The toxicity of the compounds of the present invention is very low and therefore, it may be considered that the compounds of the present invention are safe for pharmaceutical use.
For the purpose above described, the compounds of the present invention of the formula (I), non-toxic salts and acid addition salts thereof and hydrates thereof may be normally administered systemically or locally, usually by oral or parenteral administration.
The doses to be administered are determined depending upon age, body weight, symptom, the desired therapeutic effect, the route of administration, and the duration of the treatment etc. In the human adult, the doses per person per dose are generally between 1 mg and 1000 mg, by oral administration, up to several times per day, and between 0.1 mg and 100 mg, by parenteral administration (preferred into vein) up to several times per day, or continuous administration between 1 and 24 hrs. per day into vein.
As mentioned above, the doses to be used depend upon various conditions. Therefore, there are cases in which doses lower than or greater than the ranges specified above may be used.
The compounds of the present invention may be administered as inner solid compositions or inner liquid compositions for oral administration, or as injections, liniments or suppositories etc. for parenteral administration.
Inner solid compositions for oral administration include compressed tablets, pills, capsules, dispersible powders and granules etc. Capsules contain hard capsules and soft capsules.
In such inner solid compositions, one or more of the active compound(s) is or are, admixed with at least one inert diluent (lactose, mannitol, glucose, microcrystalline cellulose, starch etc.), connecting agents (hydroxypropyl cellulose, polyvinylpyrrolidone, magnesium metasilicate aluminate etc.), disintegrating a, agents (cellulose calcium glycolate etc.), lubricating agents (magnesium stearate etc.), stabilizing agents, assisting agents for dissolving (glutamic acid, asparaginic acid etc.) etc. to prepare pharmaceuticals by known methods. The pharmaceuticals may, if desired, be coated with material such as sugar, gelatin, hydroxypropyl cellulose or hydroxypropyl cellulose phthalate etc., or be coated with two or more films. And further, coating may include containment within capsules of absorbable materials such as gelatin.
Inner liquid compositions for oral administration include pharmaceutically-acceptable water-agents, suspensions, emulsions, syrups and elixirs etc. In such liquid compositions, one or more of the active compound(s) is or are comprised in inert diluent(s) commonly used in the art (purified water, ethanol or mixture thereof etc.). Besides inert diluents, such compositions may also comprise adjuvants such as wetting agents, suspending agents, emulsifying agents, sweetening agents, flavoring agents, perfuming agents, preserving agents and buffer agents etc.
Injections for parenteral administration include solutions, suspensions and emulsions and solid injections which are dissolved or suspended in solvent when it is used. One or more active compound(s) is or are dissolved, suspended or emulsified in a solvent when such compositions are used. Aqueous solutions or suspensions include distilled water for injection and physiological salt solution plant oil, propylene glycol, polyethylene glycol and alcohol such as ethanol etc., and mixture thereof. Such compositions may comprise additional diluents such as stabilizing agent, assisting agents for dissolving (glutamic acid, asparaginic acid, POLYSOLBATE 80 (registered trade mark) etc.), suspending agents, emulsifying agents, dispersing agents, buffer agents, preserving agents etc. They may be sterilized for example, by filtration through a bacteria-retaining filter, by incorporation of sterilizing agents in the compositions or by irradiation. They may also be manufactured in the form of sterile solid compositions and which can be dissolved in sterile water or some other sterile diluent for injection immediately before use.
Other compositions for parenteral administration include liquids for external use, ointments, endermic liniments, aerosols, spray compositions, suppositories and pessaries for vaginal administration etc. which comprise one or more of the active compound(s) and may be prepared by known methods.
Spray compositions may comprise additional substances other than inert diluents: e.g. stabilizing agents such as sodium hydrogen sulfate, stabilizing agents to give isotonicity, isotonic buffer such as sodium chloride, sodium citrate, citric acid. For preparation of such spray compositions, for example, the method described in the U.S. Pat. No. 2,868,691 or 3,095,355 may be used.